Pathogenic bacteria must assemble and secrete virulence factors in order to interact with their hosts and cause disease. Gram-negative bacteria have an outer membrane in addition to a cytoplasmic membrane and must secrete virulence factors across both these barriers. The mechanisms by which this occurs can be quite complex and are not well understood. The chaperone/usher pathway is a virulence protein secretion pathway that requires two components for secretion across the outer membrane: a periplasmic chaperone and an outer membrane protein termed an usher. The chaperone directs proper folding of the secreted proteins and prevents their engagement in non-productive interactions. The usher serves as an assembly platform at the outer membrane and provides a secretion channel to the cell surface. The chaperone/usher pathway is required for assembly and secretion of a superfamily of adhesive structures in a broad range of Gram-negative pathogens. The prototypical organelles assembled by this pathway are the P and type 1 pili expressed by uropathogenic Escherichia coli, the primary causative agent of urinary tract infections. P and type 1 pili are critical virulence factors, allowing binding and colonization of the kidney and bladder, respectively. The long-term goal of this proposal is to use pilus biogenesis by uropathogenic E. coli as a model system with which to understand virulence factor secretion in Gram-negative bacteria. More specifically, the structure and function of the usher will be investigated to elucidate the molecular mechanisms governing secretion across the outer membrane. The first specific aim is to create a detailed model of the structural arrangement of the usher in the outer membrane using computer analysis and epitope mapping techniques. The second specific aim is to probe function of the usher through generation and analysis of mutants. The third specific aim is to establish a cell-free system for pilus biogenesis based on reconstitution of the usher into liposomes. Such a system will provide an invaluable tool for studying the chaperone/usher pathway and analyzing mutants. The work described in this proposal will elucidate mechanisms of virulence factor secretion and create opportunities for the development of novel antimicrobial agents to treat not only urinary tract infections, but also a broad range of infectious diseases.